Novel human MLF3 protein

ABSTRACT

The present invention provides a novel human integral membrane (MLF3) and polynucleotides which identify and encode MLF3. The invention also provides genetically engineered expression vectors and host cells comprising the nucleic acid sequences encoding MLF3 and a method for producing MLF3. The invention also provides for agonists, antibodies, or antagonists specifically binding MLF3, and their use, in the prevention and treatment of diseases associated with expression of MLF3. Additionally, the invention provides for the use of antisense molecules to polynucleotides encoding MLF3 for the treatment of diseases associated with the expression of MLF3. The invention also provides diagnostic assays which utilize the polynucleotide, or fragments or the complement thereof, and antibodies specifically binding MLF3.

[0001] This application is a continuation of U.S. application Ser. No.09/205,449, filed Dec. 4, 1998, which is a divisional of U.S.application Ser. No. 08/805,965, filed Feb. 25, 1997, now U.S. Pat. No.5,851,774, issued Dec. 22, 1998, both entitled NOVEL HUMAN MLF3 PROTEIN,all of which applications and patents are hereby expressly incorporatedby reference herein.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acid and amino acid sequencesof a novel human protein, MLF3, and to the use of these sequences in thediagnosis, prevention, and treatment of disease.

BACKGROUND OF THE INVENTION

[0003] The MLF (myelodysplasia/myeloid leukemia factor) gene familycomprises the MLF1 and MLF2 genes which encode cytoplasmic proteins.Rearrangement of the MLF1 gene is observed in patients withmyelodysplastic syndrome and acute myeloid leukemia (AML);myelodysplastic syndrome precedes AML in certain cases. Thet(3:5)(q25.1;q34) chromosomal translocation, a non-random translocationwhich appears to be restricted to myelodysplastic syndrome and AML,creates a fusion between sequences of the nucleophosmin (NPM) gene andthe MLF1 gene. This rearrangement results in the expression of a fusionprotein whose amino-terminal end contains most of the structural motifsof the NPM protein, including a nuclear localization signal, fused toresidues 17-268 of the MLF1 protein. The NPM-MLF1 fusion proteinlocalizes to the nucleus, with highest levels seen in the nucleolus[Yoneda-Kato, N. et al. (1996) Oncogene 12:265]. Identical fusionjunctions were found in three AML patients suggesting that the NPM-MLF1fusion protein is involved in the events that lead to the clonaldisruption of hematopoietic cell development characteristic of themyelodysplastic and leukemic phases of AML (Yoneda-Kato, N. et al.,supra). The MLF1 gene is normally expressed in a variety of tissues withthe highest levels of expression seen in testis, ovary, skeletal andcardiac muscle, colon and kidney. Low levels or no expression of MLF1 isseen in spleen, thymus and peripheral blood leukocytes.

[0004] The MFL2 gene encodes a protein which is highly related (40%identity, 63% similarity) to the MLF1 protein (Kuefer, M. U. et al.(1996) Genomics 35:392). The MFL2 gene is ubiquitously expressed incontrast to the tissue-restricted pattern of expression displayed by theMLF1 gene. The MFL2 gene maps to human chromosome 12p13, a regionfrequently involved in deletions and translocations in acute myeloid andlymphoid leukemias (Kuefer, M. U. et al., supra).

[0005] The discovery of molecules related to the MLF gene familysatisfies a need in the art by providing new diagnostic or therapeuticcompositions useful in the treatment of disorders associated withalterations in the expression of members of the MLF gene family.

SUMMARY OF THE INVENTION

[0006] The present invention features a novel protein hereinafterdesignated MLF3 and characterized as having similarity to the human MLF1and MFL2 proteins.

[0007] Accordingly, the invention features a substantially purifiedpolypeptide having the amino acid sequence shown in SEQ ID NO:1 orfragments thereof. Preferred fragments of SEQ ID NO:1 are fragments ofabout 15 amino acids or greater in length which define fragments unique(i.e., having less than about 25% identity to fragments of anotherprotein) to SEQ ID NO:1 or which retain biological activity orimmunological activity (i.e., capable of eliciting anti-MLF3antibodies). Fragments of SEQ ID NO:1 which are at least 25 amino acids,at least 50 amino acids, at least 100 amino acids, at least 125 aminoacids, at least 200 amino acids and at least 250 amino acids in lengthare contemplated.

[0008] The present invention further provides isolated and substantiallypurified polynucleotide sequences encoding the polypeptide comprisingthe amino acid sequence of SEQ ID NO:1 or fragments thereof. In aparticular aspect, the polynucleotide is the nucleotide sequence of SEQID NO:2 or variants thereof. In another embodiment, the presentinvention provides polynucleotides comprising fragments of SEQ ID NO:2having a length greater than 20 nucleotides. The invention furthercontemplates fragments of this polynucleotide sequence (i.e., SEQ IDNO:2) that are at least 50 nucleotides, at least 100 nucleotides, atleast 250 nucleotides, at least 500 nucleotides and at least 950nucleotides in length.

[0009] In addition, the invention provides polynucleotide sequenceswhich hybridize under stringent conditions to the polynucleotidesequence of SEQ ID NO:2. In another embodiment the present inventionprovides a composition comprising an isolated and purifiedpolynucleotide sequence encoding MLF3.

[0010] The invention provides polynucleotide sequences comprising thecomplement of SEQ ID NO:2 or variants thereof; these complementarynucleic acid sequences may comprise the complement of the entire nucleicacid sequence of SEQ ID NO:2 or fragments thereof. In another embodimentthe present invention provides a composition comprising an isolated andpurified polynucleotide sequence comprising the complement of SEQ IDNO:2 or variants thereof.

[0011] The invention additionally features nucleic acid sequencesencoding polypeptides, oligonucleotides, peptide nucleic acids (PNA),fragments, portions or antisense molecules thereof, and expressionvectors and host cells comprising polynucleotides that encode MLF3.

[0012] In another embodiment the present invention provides an isolatedpolynucleotide comprising at least a portion of the nucleic acidsequence of SEQ ID NO:2 or variants thereof contained on a recombinantexpression vector. In yet another embodiment, the expression vectorcontaining the polynucleotide sequence is contained within a host cell.The invention is not limited by the nature of the host cell employed.For example, the host cell may be an E. coli cell, a yeast cell, aninsect cell, a mammalian cell, etc.

[0013] The present invention also provides a method for producing apolypeptide comprising the amino acid sequence of SEQ ID NO:1 orfragments thereof, the method comprising the steps of: a) culturing thehost cell containing an expression vector containing an isolatedpolynucleotide encoding at least a fragment of the MLF3 polypeptideunder conditions suitable for the expression of the polypeptide; and b)recovering the polypeptide from the host cell culture.

[0014] In another embodiment, the invention provides a pharmaceuticalcomposition comprising a substantially purified human MLF3 proteinhaving the amino acid sequence of SEQ ID NO:1 in conjunction with asuitable pharmaceutical carrier.

[0015] The invention also provides a purified antibody which bindsspecifically to a polypeptide comprising at least a portion of the aminoacid sequence of SEQ ID NO:1.

[0016] Still further, the invention provides a purified agonist whichspecifically binds to and modulates the activity of a polypeptidecomprising at least a portion of the amino acid sequence of SEQ ID NO:1.The present invention further provides a pharmaceutical compositioncomprising a purified agonist which specifically binds to and modulatesthe activity of a polypeptide comprising at least a portion of the aminoacid sequence of SEQ ID NO:1. In another embodiment, the inventionprovides a purified antagonist which specifically binds to and modulatesthe activity of a polypeptide comprising at least a portion of the aminoacid sequence of SEQ ID NO:1. The present invention further provides apharmaceutical composition comprising a purified antagonist whichspecifically binds to and modulates the activity of a polypeptidecomprising at least a portion of the amino acid sequence of SEQ ID NO:1.

[0017] The invention also provides a method for treating breast cancercomprising administering to a subject in need of such treatment aneffective amount of a pharmaceutical composition comprising a purifiedantagonist which specifically binds to and modulates the activity of apolypeptide comprising at least a portion of the amino acid sequence ofSEQ ID NO:1. The treatment of a variety of tumors, including but notlimited to brain, stomach, paraganglionic tumors, using agonists as wellas antagonists of MLF3 is also contemplated by the present invention.

[0018] The invention also provides a method for the detection ofpolynucleotides encoding human MLF3 in a biological sample comprisingthe steps of: a) hybridizing a polynucleotide sequence encoding humanMLF3 (SEQ ID NO:1) to nucleic acid material of a biological sample,thereby forming a hybridization complex; and b) detecting thehybridization complex, wherein the presence of the complex correlateswith the presence of a polynucleotide encoding human MLF3 in thebiological sample. In a preferred embodiment, prior to hybridization,the nucleic acid material of the biological sample is amplified by thepolymerase chain reaction. In another preferred embodiment, the nucleicacid material comprises metaphase chromosomes prepared from human cells(e.g., from a biopsy or blood sample) and detection of the hybridizationcomplex indicates the chromosomal location (i.e., normal or rearranged)of the MLF3 gene in the human cells.

BRIEF DESCRIPTION OF THE FIGURES

[0019]FIGS. 1A and 1B show the amino acid sequence (SEQ ID NO:1) andnucleic acid sequence (SEQ ID NO:2) of MLF3. The alignment was producedusing MACDNASIS PRO™ software (Hitachi Software Engineering Co., Ltd.,San Bruno, Calif.).

[0020]FIG. 2 shows the amino acid sequence alignments among MLF3 (SEQ IDNO:1) MLF1 (GI 1066392: SEQ ID NO:3) and MLF2 (GI 1399745; SEQ ID NO:5).The alignment was produced using the multisequence alignment program ofDNASTAR™ software (DNASTAR Inc, Madison Wis.).

[0021]FIGS. 3A, 3B and 3C show the hydrophobicity plots (MACDNASIS PRO™software) for MLF3 (SEQ ID NO:1), MLF2 (SEQ ID NO:5), and MLF1 (SEQ IDNO:3), respectively. The positive X axis reflects amino acid position,and the negative Y axis, hydrophobicity.

[0022]FIGS. 4A and 4B show the northern analysis for SEQ ID NO:1. Thenorthern analysis was produced electronically using LIFESEQ-FL™ database(Incyte Pharmaceuticals, Inc., Palo Alto, Calif.).

DESCRIPTION OF THE INVENTION

[0023] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0024] It must be noted that, as used herein, and in the appendedclaims, the singular forms “a”, “an”, and “the” include plural referenceunless the context clearly dictates otherwise. Thus, for example,reference to “a host cell” includes a plurality of such host cells,reference to the “antibody” is a reference to one or more antibodies andequivalents thereof known to those skilled in the art, and so forth.

[0025] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publicationsmentioned herein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

Definitions

[0026] “Nucleic acid sequence” as used herein refers to anoligonucleotide, nucleotide, or polynucleotide, and fragments orportions thereof, and to DNA or RNA of genomic or synthetic origin whichmay be single- or double-stranded, and represent the sense or antisensestrand. Similarly, “amino acid sequence” as used herein refers to anoligopeptide, peptide, polypeptide, or protein sequence, and fragmentsor portions thereof, and to naturally occurring or synthetic molecules.

[0027] A “composition comprising a given polynucleotide sequence” asused herein refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise an aqueoussolution. Compositions comprising polynucleotide sequences encoding MLF3(SEQ ID NO:1) or fragments thereof (e.g., SEQ ID NO:2 and fragmentsthereof) may be employed as hybridization probes. In this case, theMLF3-encoding polynucleotide sequences are typically employed in anaqueous solution containing salts (e.g., NaCl), detergents (e.g., SDS)and other components (e.g., Denhardt's solution, dry milk, salmon spermDNA, etc.).

[0028] Where “amino acid sequence” is recited herein to refer to anamino acid sequence of a naturally occurring protein molecule, “aminoacid sequence” and like terms, such as “polypeptide” or “protein” arenot meant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

[0029] “Peptide nucleic acid”, as used herein, refers to a moleculewhich comprises an oligomer to which an amino acid residue, such aslysine, and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary strand of nucleic acid (Nielsen, P. E. et al. (1993)Anticancer Drug Des. 8:53-63).

[0030] MLF3, as used herein, refers to the amino acid sequences ofsubstantially purified MLF3 obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

[0031] “Consensus”, as used herein, refers to a nucleic acid sequencewhich has been resequenced to resolve uncalled bases, or which has beenextended using XL-PCR™ (Perkin Elmer, Norwalk, Conn.) in the 5′ and/orthe 3′ direction and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte clone using the GELVIEW™Fragment Assembly system (GCG, Madison, Wis.), or which has been bothextended and assembled.

[0032] A “variant” of MLF3, as used herein, refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant may have “nonconservative”changes, e.g., replacement of a glycine with a tryptophan. Similar minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

[0033] A “deletion”, as used herein, refers to a change in either aminoacid or nucleotide sequence in which one or more amino acid ornucleotide residues, respectively, are absent.

[0034] An “insertion” or “addition”, as used herein, refers to a changein an amino acid or nucleotide sequence resulting in the addition of oneor more amino acid or nucleotide residues, respectively, as compared tothe naturally occurring molecule.

[0035] A “substitution”, as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively.

[0036] The term “biologically active”, as used herein, refers to aprotein having structural, regulatory, or biochemical functions of anaturally occurring molecule. Likewise, “immunologically active” refersto the capability of the natural, recombinant, or synthetic MLF3, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0037] The term “agonist”, as used herein, refers to a molecule which,when bound to MLF3, causes a change in MLF3 which modulates the activityof MLF3. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to MLF3.

[0038] The terms “antagonist” or “inhibitor”, as used herein, refer to amolecule which, when bound to MLF3, blocks or modulates the biologicalor immunological activity of MLF3. Antagonists and inhibitors mayinclude proteins, nucleic acids, carbohydrates, or any other moleculeswhich bind to MLF3.

[0039] The term “modulate”, as used herein, refers to a change or analteration in the biological activity of MLF3. Modulation may be anincrease or a decrease in protein activity, a change in bindingcharacteristics, or any other change in the biological, functional, orimmunological properties of MLF3.

[0040] The term “mimetic”, as used herein, refers to a molecule, thestructure of which is developed from knowledge of the structure of MLF3or portions thereof and, as such, is able to effect some or all of theactions of MLF3-like molecules.

[0041] The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding MLF3 or the encoded MLF3.Illustrative of such modifications would be replacement of hydrogen byan alkyl, acyl, or amino group. A nucleic acid derivative would encode apolypeptide which retains essential biological characteristics of thenatural molecule.

[0042] The term “substantially purified”, as used herein, refers tonucleic or amino acid sequences that are removed from their naturalenvironment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated.

[0043] “Amplification”, as used herein, refers to the production ofadditional copies of a nucleic acid sequence and is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, aLaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

[0044] The term “hybridization”, as used herein, refers to any processby which a strand of nucleic acid bonds with a complementary strandthrough base pairing.

[0045] The term “hybridization complex”, as used herein, refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen binds between complementary G and C bases andbetween complementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which cells have beenfixed for in situ hybridization).

[0046] The terms “complementary” or “complementarity”, as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A”. Complementaritybetween two single-stranded molecules may be “partial”, in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands.

[0047] The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding, the probe will not hybridize tothe second non-complementary target sequence.

[0048] As known in the art, numerous equivalent conditions may beemployed to comprise either low or high stringency conditions. Factorssuch as the length and nature (DNA, RNA, base composition) of thesequence, nature of the target (DNA, RNA, base composition, presence insolution or immobilization, etc.), and the concentration of the saltsand other components (e.g., the presence or absence of formamide,dextran sulfate and/or polyethylene glycol) are considered and thehybridization solution may be varied to generate conditions of eitherlow or high stringency different from, but equivalent to, the abovelisted conditions.

[0049] The term “stringent conditions”, as used herein, is the“stringency” which occurs within a range from about Tm-5° C. (5° C.below the melting temperature (Tm) of the probe) to about 20° C. to 25°C. below Tm. As will be understood by those of skill in the art, thestringency of hybridization may be altered in order to identify ordetect identical or related polynucleotide sequences. Under “stringentconditions” SEQ ID NO:2 or fragments thereof will hybridize to its exactcomplement and closely related sequences. The stringent conditions arechosen such that SEQ ID NO:2 or fragments thereof will hybridize tosequences encoding human MLF3 but not to sequences encoding human MLF1(i.e., SEQ ID NO:4 or its RNA equivalents) or MLF2 (SEQ ID NO:6 or itsRNA equivalents). When fragments of SEQ ID NO:2 are employed inhybridization reactions, the stringent conditions include the choice offragments of SEQ ID NO:2 to be used. Fragments of SEQ ID NO:2 whichcontain unique sequences (i.e., regions which are either non-homologousto or which contain less than about 50% homology or complementarity withSEQ ID NOS:4 and 6) are preferentially employed. SEQ ID NO:4 representsDNA sequences encoding the human MLF1 protein; this DNA sequence can befound in GenBank under accession number 1066391. SEQ ID NO:6 representsDNA sequences encoding the human MLF2 protein; this DNA sequence can befound in GenBank under accession number 1399744.

[0050] The term “antisense”, as used herein, refers to nucleotidesequences which are complementary to a specific DNA or RNA sequence. Theterm “antisense strand” is used in reference to a nucleic acid strandthat is complementary to the “sense” strand. Antisense molecules may beproduced by any method, including synthesis by ligating the gene(s) ofinterest in a reverse orientation to a viral promoter which permits thesynthesis of a complementary strand. Once introduced into a cell, thistranscribed strand combines with natural sequences produced by the cellto form duplexes. These duplexes then block either the furthertranscription or translation. In this manner, mutant phenotypes may begenerated. The designation “negative” is sometimes used in reference tothe antisense strand, and “positive” is sometimes used in reference tothe sense strand.

[0051] The term “portion”, as used herein, with regard to a protein (asin “a portion of a given protein”) refers to fragments of that protein.The fragments may range in size from four amino acid residues to theentire amino acid sequence minus one amino acid. Thus, a protein“comprising at least a portion of the amino acid sequence of SEQ IDNO:1” encompasses the full-length human MLF3 and fragments thereof.

[0052] “Transformation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the host cell being transformedand may include, but is not limited to, viral infection,electroporation, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

[0053] The term “antigenic determinant”, as used herein, refers to thatportion of a molecule that makes contact with a particular antibody(i.e., an epitope). When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

[0054] The terms “specific binding” or “specifically binding”, as usedherein, in reference to the interaction of an antibody and a protein orpeptide, mean that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words, the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A”, the presence of aprotein containing epitope A (or free, unlabeled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

[0055] The term “sample”, as used herein, is used in its broadest sense.A biological sample suspected of containing nucleic acid encoding MLF3or fragments thereof may comprise a cell, chromosomes isolated from acell (e.g., a spread of metaphase chromosomes), genomic DNA (in solutionor bound to a solid support such as for Southern analysis), RNA (insolution or bound to a solid support such as for northern analysis),cDNA (in solution or bound to a solid support), an extract from cells ora tissue, and the like.

[0056] The term “correlates with expression of a polynucleotide”, asused herein, indicates that the detection of the presence of ribonucleicacid that is similar to SEQ ID NO:2 by northern analysis is indicativeof the presence of mRNA encoding MLF3 in a sample and thereby correlateswith expression of the transcript from the polynucleotide encoding theprotein. “Alterations” in the polynucleotide of SEQ ID NO:2, as usedherein, comprise any alteration in the sequence of polynucleotidesencoding MLF3 including deletions, insertions, and point mutations thatmay be detected using hybridization assays. Included within thisdefinition is the detection of alterations to the genomic DNA sequencewhich encodes MLF3 (e.g., by alterations in the pattern of restrictionfragment length polymorphisms capable of hybridizing to SEQ ID NO:2),the inability of a selected fragment of SEQ ID NO:2 to hybridize to asample of genomic DNA (e.g., using allele-specific oligonucleotideprobes), and improper or unexpected hybridization, such as hybridizationto a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding MLF3 (e.g., using fluorescent in situhybridization [FISH] to metaphase chromosomes spreads).

[0057] As used herein, the term “antibody” refers to intact molecules aswell as fragments thereof, such as Fa, F(ab′)₂, and Fv, which arecapable of binding the epitopic determinant. Antibodies that bind MLF3polypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or peptide used to immunize an animal can be derived fromthe transition of RNA or synthesized chemically, and can be conjugatedto a carrier protein, if desired. Commonly used carriers that arechemically coupled to peptides include bovine serum albumin andthyroglobulin. The coupled peptide is then used to immunize the animal(e.g., a mouse, a rat, or a rabbit).

[0058] The term “humanized antibody”, as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

The Invention

[0059] The invention is based on the discovery of a novel human protein(MLF3), the polynucleotides encoding MLF3, and the use of thesecompositions for the diagnosis, prevention, or treatment of diseasesassociated with altered or abnormal MLF3 expression. As mRNA encodingMLF3 is found in a number of tumors, MLF3 serves as a marker forcancerous cells, particularly breast, brain, stomach and paraganglionictumor cells. In addition, MLF3 is expressed at the highest levels inbrain and therefore serves as a marker for brain tissue.

[0060] Nucleic acids encoding the human MLF3 of the present inventionwere first identified in Incyte Clone 762280 from the BRAITUT02 cDNAlibrary through a computer-generated search for amino acid sequencealignments. A consensus sequence, SEQ ID NO:2, was derived from thefollowing overlapping and/or extended nucleic acid sequences: IncyteClones 762280 (BRAITUT02), 912942 (STOMNOT02), 896085 (BRSTNOT05),892034 (STOMTUT01), 774094 (COLONNOT05), 728689 (LUNGNOT03), 720563(SYNOOAT01), 644216 (BRSTTUT02), 620396 (PGANNOT01), 1291517(BRAINOT11), 1266914 (BRAINOT09), 1261396 (SYNORAT05), 1253043(LUNGFET03) and 1219407 (NEUTGMT01).

[0061] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1Aand 1B. MLF3 is 262 amino acids in length and contains a single cysteineresidue (i.e., C₉₉). In addition to providing sites for disulfide bondformation, cysteine residues provide potential sites for palmitoylation.The human MLF3 of the present invention contains sequences which closelymatch an aminoacyl-transfer RNA synthetases class-II signature motif[Schimmel P (1987) Annu. Rev. Biochem. 56:125] (i.e., residues 192-215and 196-215 of SEQ ID NO:1). MLF3 contains a sequence (i.e., residues71-75) which closely matches the serine active site consensus sequenceGDSGG found in serine proteases from the trypsin family [Brenner, S.(1988) Nature 334:528]. MLF3 contains a potential C-terminal amidationsite (i.e., residues 220-223), a potential cAMP- and cGMP-dependentprotein kinase phosphorylation site (i.e., residues 197-200), fourpotential casein kinase II phosphorylation sites (i.e., residues 42-45,140-143, 146-149 and 238-241) and four potential protein kinase Cphosphorylation sites (i.e., residues 55-57, 137-139, 140-142 and200-202). MLF3 contains a potential glycosaminoglycan attachment site(i.e., residues 217-220) and 6 potential N-myristoylation sites (i.e.,residues 46-51, 50-55, 71-76, 97-102, 133-138 and 220-225); these sitesare located internally to MLF3 and therefore proteolytic processingwhich exposes an internal glycine would be a prerequisite toN-myristoylation of MLF3.

[0062] MFL3 contains a stretch of amino acid residues (residues 118-199of SEQ ID NO:1) which share homology (27% identity and 43% similarity)with the Trichomonas vaginalis cysteine protease (TVCYSP) which isproposed to play a role in the pathogenesis of T. vaginalis infection(Garber, G. E. et al. (1993) Appl. Parasitol. 34:245). Homology toTVCYSP is also seen in MLF1 (residues 121-202 of SEQ ID NO:3) and MLF2(residues 118-199 of SEQ ID NO:5) (Kuefer, M. U. et al., supra).

[0063] MLF3 contains a stretch of 35 amino acid residues (residues128-162 of SEQ ID NO:1) which is highly conserved between MLF1 and MLF2and has been proposed to represent a motif of functional importance(Kuefer, M. U. et al., supra).

[0064] The MLF3 protein of the present invention, like the human MLF2protein, has an acidic isoelectric point (pI) (MLF3 has a pI of 6.0 andMLF2 has a pI of 6.45). In addition, the MLF3 protein of the presentinvention, like the human MLF1 and MLF2 proteins, has a high content ofarginine residues (MLF3 contains 11.5% arginine; MLF2 contains 12.5%arginine; MLF1 contains 9% arginine). As illustrated by FIGS. 3A, 3B and3C, MLF3, MLF1 and MLF2 have similar hydrophobicity plots, with theplots of MLF3 (FIG. 3A) and MLF2 (FIG. 3B) showing the most similarity.

[0065] MLF3 has chemical and structural homology with the human MLF1 (GI1066392; SEQ ID NO:3) and MLF2 (GI 1399745; SEQ ID NO:5) proteins(Yoneda-Kato, N. et al., supra and Kuefer, M. U. et al., supra). Inparticular, MLF3 and MLF1 share 35% identity and 53% similarity and MLF3and MLF2 share 89% identity. A pair of residues are said to be similarif they represent conservative substitutions. FIG. 2 provides analignment between the amino acid sequences of SEQ ID NOS:1,3 and 5.

[0066] Northern analysis (FIGS. 4A and 4B) shows the expression ofMLF3-encoding sequences in various libraries, at least 29% of which arecancerous or immortalized and at least 12% of which are involved withthe hematopoietic system and/or immune response, including inflammatoryand/or autoimmune disease (e.g., Crohn's disease). Of particular note isthe expression of MLF3 mRNA in breast tumor (3/77), brain tumor (2/77),stomach tumor (2/77) and paraganglionic tumor (2/77) libraries. Thispattern of expression demonstrates that MLF3 serves as a marker forcancerous cells, particularly breast tumor cells. In addition to itsexpression in a variety of tumors, MLF3 is highly expressed in brain andthus serves as a marker for this tissue. At least 15% of the librariescontaining sequences derived from MLF3 mRNA are derived from eithernormal or diseased brain tissue and at least 11% of the librariescontaining MLF3 sequences are derived from cells or tissues in thehematopoietic lineages.

[0067] The invention also encompasses MLF3 variants. A preferred MLF3variant is one having at least 80%, and more preferably 90%, amino acidsequence similarity to the MLF3 amino acid sequence (SEQ ID NO:1). Amost preferred MLF3 variant is one having at least 95% amino acidsequence similarity to SEQ ID NO:1.

[0068] The invention also encompasses polynucleotides which encode MLF3.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of MLF3 can be used to generate recombinant molecules whichexpress MLF3. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid sequence of SEQ ID NO:2 asshown in FIGS. 1A and 1B.

[0069] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of nucleotidesequences encoding MLF3, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.Thus, the invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring MLF3, and all such variations are to beconsidered as being specifically disclosed.

[0070] Although nucleotide sequences which encode MLF3 and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring MLF3 under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding MLF3 or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding MLF3 and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

[0071] The invention also encompasses production of DNA sequences, orportions thereof, which encode MLF3 and its derivatives, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents that are well known in the art at the time of thefiling of this application. Moreover, synthetic chemistry may be used tointroduce mutations into a sequence encoding MLF3 or any portionthereof.

[0072] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed nucleotide sequences, andin particular, those shown in SEQ ID NO:2, under various conditions ofstringency. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe, as taughtin Wahl, G. M. and S. L. Berger (1987; Methods Enzymol. 152:399-407) andKimmel, A. R. (1987; Methods Enzymol. 152:507-511), and may be used at adefined stringency.

[0073] Altered nucleic acid sequences encoding MLF3 which areencompassed by the invention include deletions, insertions, orsubstitutions of different nucleotides resulting in a polynucleotidethat encodes the same or a functionally equivalent MLF3. The encodedprotein may also contain deletions, insertions, or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent MLF3. Deliberate amino acid substitutions may bemade on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues as long as the biological activity of MLF3 is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid; positively charged amino acids may include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline; glycine and alanine; asparagine and glutamine; serine andthreonine; phenylalanine and tyrosine.

[0074] Also included within the scope of the present invention arealleles of the genes encoding MLF3. As used herein, an “allele” or“allelic sequence” is an alternative form of the gene which may resultfrom at least one mutation in the nucleic acid sequence. Alleles mayresult in altered mRNAs or polypeptides whose structure or function mayor may not be altered. Any given gene may have none, one, or manyallelic forms. Common mutational changes which give rise to alleles aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

[0075] Methods for DNA sequencing which are well known and generallyavailable in the art may be used to practice any embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, SEQUENASE® (US Biochemical Corp, Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of recombinant polymerases andproofreading exonucleases such as the ELONGASE Amplification Systemmarketed by Gibco BRL (Gaithersburg, Md.). Preferably, the process isautomated with machines such as the Hamilton MICRO LAB 2200 (Hamilton,Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown,Mass.) and the ABI 377 DNA sequencers (Perkin Elmer).

[0076] The nucleic acid sequences encoding MLF3 may be extendedutilizing a partial nucleotide sequence and employing various methodsknown in the art to detect upstream sequences such as promoters andregulatory elements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to linker sequence and a primer specific to the knownregion. The amplified sequences are then subjected to a second round ofPCR with the same linker primer and another specific primer internal tothe first one. Products of each round of PCR are transcribed with anappropriate RNA polymerase and sequenced using reverse transcriptase.

[0077] Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed using OLIGO4.06 Primer Analysis software (National Biosciences Inc., Plymouth,Minn.), or another appropriate program, to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68°-72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

[0078] Another method which may be used is capture PCR which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1: 111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown portion of the DNA moleculebefore performing PCR.

[0079] Another method which may be used to retrieve unknown sequences isthat of Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTERFINDER™libraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

[0080] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into the 5′ and 3′non-transcribed regulatory regions.

[0081] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled device camera. Output/light intensity may be converted toelectrical signal using appropriate software (e.g. GENOTYPER™ andSEQUENCE NAVIGATOR™, Perkin Elmer) and the entire process from loadingof samples to computer analysis and electronic data display may becomputer controlled. Capillary electrophoresis is especially preferablefor the sequencing of small pieces of DNA which might be present inlimited amounts in a particular sample.

[0082] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode MLF3, or fusion proteins or functionalequivalents thereof, may be used in recombinant DNA molecules to directexpression of MLF3 in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and these sequences may be used to clone and expressMLF3.

[0083] As will be understood by those of skill in the art, it may beadvantageous to produce MLF3-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce a recombinant RNAtranscript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence.

[0084] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterMLF3 encoding sequences for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, or introduce mutations, and so forth.

[0085] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding MLF3 may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of MLF3 activity, it may be useful toencode a chimeric MLF3 protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the MLF3 encoding sequence and theheterologous protein sequence, so that MLF3 may be cleaved and purifiedaway from the heterologous moiety.

[0086] In another embodiment, sequences encoding MLF3 may besynthesized, in whole or in part, using chemical methods well known inthe art (see Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of MLF3, or a portion thereof. Forexample, peptide synthesis can be performed using various solid-phasetechniques (Roberge, J. Y. et al. (1995) Science 269:202-204) andautomated synthesis may be achieved, for example, using the ABI 431APeptide Synthesizer (Perkin Elmer).

[0087] The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, WH Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of MLF3, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0088] In order to express a biologically active MLF3, the nucleotidesequences encoding MLF3 or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

[0089] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encoding MLF3and appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

[0090] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding MLF3. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

[0091] The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector--enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT® phagemid (Stratagene,LaJolla, Calif.) or PSPORT1™ plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding MLF3,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

[0092] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for MLF3. For example, whenlarge quantities of MLF3 are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be used. Such vectors include, but are not limitedto, the multifunctional E. coli cloning and expression vectors such asBLUESCRIPT® (Stratagene), in which the sequence encoding MLF3 may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

[0093] In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.(supra) and Grant et al. (1987) Methods Enzymol. 153:516-544. In caseswhere plant expression vectors are used, the expression of sequencesencoding MLF3 may be driven by any of a number of promoters. Forexample, viral promoters such as the 35S and 19S promoters of CaMV maybe used alone or in combination with the omega leader sequence from TMV(Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plant promoterssuch as the small subunit of RUBISCO or heat shock promoters may be used(Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al.(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl.Cell Differ. 17:85-105). These constructs can be introduced into plantcells by direct DNA transformation or pathogen-mediated transfection.Such techniques are described in a number of generally available reviews(see, for example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook ofScience and Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196.

[0094] An insect system may also be used to express MLF3. For example,in one such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encoding MLF3may be cloned into a non-essential region of the virus, such as thepolyhedrin gene, and placed under control of the polyhedrin promoter.Successful insertion of MLF3 will render the polyhedrin gene inactiveand produce recombinant virus lacking coat protein. The recombinantviruses may then be used to infect, for example, S. frugiperda cells orTrichoplusia larvae in which MLF3 may be expressed (Engelhard, E. K. etal. (1994) Proc. Natl. Acad. Sci. 91:3224-3227).

[0095] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, sequences encoding MLF3 may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable viruswhich is capable of expressing MLF3 in infected host cells (Logan, J.and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0096] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding MLF3. Such signals includethe ATG initiation codon and adjacent sequences. In cases wheresequences encoding MLF3, its initiation codon, and upstream sequencesare inserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a portion thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

[0097] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO (ATCC CCL 61 and CRL 9618),HeLa (ATCC CCL 2), MDCK (ATCC CCL 34 and CRL 6253), HEK 293 (ATCC CRL1573), WI-38 (ATCC CCL 75) (ATCC: American Type Culture Collection,Rockville, Md.), which have specific cellular machinery andcharacteristic mechanisms for such post-translational activities, may bechosen to ensure the correct modification and processing of the foreignprotein.

[0098] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress MLF3 may be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for 1-2 days in an enriched media before they are switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

[0099] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980)Cell 22:817-23) genes which can be employed in tk- or aprt- cells,respectively. Also, antimetabolite, antibiotic or herbicide resistancecan be used as the basis for selection; for example, dhfr which confersresistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad.Sci. 77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

[0100] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionmay need to be confirmed. For example, if the sequence encoding MLF3 isinserted within a marker gene sequence, recombinant cells containingsequences encoding MLF3 can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding MLF3 under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

[0101] Alternatively, host cells which contain the nucleic acid sequenceencoding MLF3 and express MLF3 may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

[0102] The presence of polynucleotide sequences encoding MLF3 can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or portions or fragments of polynucleotides encoding MLF3.Nucleic acid amplification based assays involve the use ofoligonucleotides or oligomers based on the sequences encoding MLF3 todetect transformants containing DNA or RNA encoding MLF3. As used herein“oligonucleotides” or “oligomers” refer to a nucleic acid sequence of atleast about 10 nucleotides and as many as about 60 nucleotides,preferably about 15 to 30 nucleotides, and more preferably about 20-25nucleotides, which can be used as a probe or amplimer.

[0103] A variety of protocols for detecting and measuring the expressionof MLF3, using either polyclonal or monoclonal antibodies specific forthe protein are known in the art. Examples include enzyme-linkedimmunosorbant assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson MLF3 is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

[0104] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding MLF3include oligolabeling, nick translation, end-labeling or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding MLF3, or any portions thereof may be cloned into a vector forthe production of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo,Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., Cleveland,Ohio). Suitable reporter molecules or labels, which may be used, includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

[0105] Host cells transformed with nucleotide sequences encoding MLF3may be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by arecombinant cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides whichencode MLF3 may be designed to contain signal sequences which directsecretion of MLF3 through a prokaryotic or eukaryotic cell membrane.

[0106] Other recombinant constructions may be used to join sequencesencoding MLF3 to nucleotide sequences encoding a polypeptide domainwhich will facilitate purification of soluble proteins. Suchpurification facilitating domains include, but are not limited to, metalchelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals, protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAGS extension/affinity purification system (Immunex Corp.,Seattle, Wash.). The inclusion of cleavable linker sequences such asthose specific for Factor XA or enterokinase (Invitrogen, San Diego,Calif.) between the purification domain and MLF3 may be used tofacilitate purification. One such expression vector provides forexpression of a fusion protein containing MLF3 and a nucleic acidencoding 6 histidine residues preceding a thioredoxin or an enterokinasecleavage site. The histidine residues facilitate purification on IMIAC(immobilized metal ion affinity chromatography) as described in Porath,J. et al. (1992, Prot. Exp. Purif. 3: 263-281) while the enterokinasecleavage site provides a means for purifying MLF3 from the fusionprotein. A discussion of vectors which contain fusion proteins isprovided in Kroll, D. J. et al. (1993; DNA Cell Biol. 12:441-453).

[0107] In addition to recombinant production, fragments of MLF3 may beproduced by direct peptide synthesis using solid-phase techniques(Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using Applied Biosystems 431APeptide Synthesizer (Perkin Elmer). Various fragments of MLF3 may bechemically synthesized separately and combined using chemical methods toproduce the full length molecule.

Therapeutics

[0108] Based on the chemical and structural homology among MLF3 (SEQ IDNO:1) and the human MLF1 and MLF2 proteins (SEQ ID NOS:3 and 5), MLF3 isa member of the MLF gene family. The rearrangement of the MLF1 gene [thet(3;5)(q25. 1;q34) translocation] is associated with myelodysplasticsyndrome and AML. The MLF2 gene maps to the short arm of humanchromosome 12 which is frequently rearranged (translocations anddeletions) in a broad spectrum of hematopoietic malignancies, includingALL, AML and myelodysplastic syndrome (Kuefer, M. U. et al., supra).Thus, chromosomal rearrangement of members of the MLF gene family areassociated with hematopoietic malignancies and therefore the MLF3 geneprovides a means to identify chromosomal rearrangements associated withhematopoietic malignancies. MLF3 is shown herein to be expressed invariety of tumors, particularly in breast, brain, stomach andparaganglionic tumors (FIGS. 4A and 4B). Furthermore, since thesequences encoding MLF3 were cloned from brain tumor tissue, MLF3expression appears to be indicative of a proliferative state.

[0109] Therefore, in one embodiment, MLF3 or a fragment or derivativethereof may be administered to a subject to treat disorders associatedwith abnormal expression of MLF3, including a variety of tumors. Suchconditions and diseases may include, but are not limited to, breast,brain, stomach and paraganglionic tumors.

[0110] In another embodiment, a vector capable of expressing MLF3, or afragment or a derivative thereof, may also be administered to a subjectto treat the breast, brain, stomach and paraganglionic tumors describedabove.

[0111] In another embodiment, MLF3 may be administered in combinationwith other conventional chemotherapeutic agents. The combination oftherapeutic agents having different mechanisms of action will havesynergistic effects allowing for the use of lower effective doses ofeach agent and lessening side effects.

[0112] In one aspect, antibodies which are specific for MLF3 may be useddirectly as an agonist, or indirectly as a targeting or deliverymechanism for bringing a pharmaceutical agent to cells or tissue whichexpress MLF3.

[0113] In one embodiment, antagonists or inhibitors of MLF3 may beadministered to a subject to treat or prevent tumors, particularlybreast, brain, stomach and paraganglionic tumors.

[0114] In another embodiment, a vector expressing antisense of thepolynucleotide encoding MLF3 may be administered to a subject to treator prevent tumors, particularly breast, brain, stomach andparaganglionic tumors.

[0115] Antagonists or inhibitors of MLF3 may be produced using methodswhich are generally known in the art. In particular, purified MLF3 maybe used to produce antibodies or to screen libraries of pharmaceuticalagents to identify those which specifically bind MLF3.

[0116] Antibodies which are specific for MLF3 may be used directly as anantagonist, or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express MLF3.The antibodies may be generated using methods that are well known in theart. Such antibodies may include, but are not limited to, polyclonal,monoclonal, chimeric, single chain, Fab fragments, and fragmentsproduced by a Fab expression library. Neutralizing antibodies, (i.e.,those which reduce or abolish MLF3 activity) are especially preferredfor therapeutic use.

[0117] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith MLF3 or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

[0118] It is preferred that the peptides, fragments, or oligopeptidesused to induce antibodies to MLF3 have an amino acid sequence consistingof at least five amino acids, and more preferably at least 10 aminoacids. It is also preferable that they are identical to a portion of theamino acid sequence of the natural protein, and they may contain theentire amino acid sequence of a small, naturally occurring molecule.Short stretches of MLF3 amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

[0119] Monoclonal antibodies to MLF3 may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120).

[0120] In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceMLF3-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobin libraries (BurtonD. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

[0121] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

[0122] Antibody fragments which contain specific binding sites for MLF3may also be generated. For example, such fragments include, but are notlimited to, the F(ab′)2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

[0123] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between MLF3 and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering MLF3 epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

[0124] In another embodiment of the invention, the polynucleotidesencoding MLF3, or any fragment thereof, or antisense molecules, may beused for therapeutic purposes. In one aspect, antisense to thepolynucleotide encoding MLF3 may be used in situations in which it wouldbe desirable to block the transcription of the mRNA. In particular,cells may be transformed with sequences complementary to polynucleotidesencoding MLF3. Thus, antisense molecules may be used to modulate MLF3activity, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligomers or largerfragments, can be designed from various locations along the coding orcontrol regions of sequences encoding MLF3.

[0125] Expression vectors derived from retroviruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisensemolecules complementary to the polynucleotides of the gene encodingMLF3. These techniques are described both in Sambrook et al. (supra) andin Ausubel et al. (supra).

[0126] Genes encoding MLF3 can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide or fragment thereof which encodes MLF3. Such constructsmay be used to introduce untranslatable sense or antisense sequencesinto a cell. Even in the absence of integration into the DNA, suchvectors may continue to transcribe RNA molecules until they are disabledby endogenous nucleases. Transient expression may last for a month ormore with a non-replicating vector and even longer if appropriatereplication elements are part of the vector system.

[0127] As mentioned above, modifications of gene expression can beobtained by designing antisense molecules, DNA, RNA, or PNA, to thecontrol regions of the gene encoding MLF3, i.e., the promoters,enhancers, and introns. Oligonucleotides derived from the transcriptioninitiation site, e.g., between positions −10 and +10 from the startsite, are preferred. Similarly, inhibition can be achieved using “triplehelix” base-pairing methodology. Triple helix pairing is useful becauseit causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature (Gee, J. E. et al. (1994) In: Huber, B.E. and B. I. Carr, Molecular and Immunologic Approaches, FuturaPublishing Co., Mt. Kisco, N.Y.). The antisense molecules may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

[0128] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding MLF3.

[0129] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0130] Antisense molecules and ribozymes of the invention may beprepared by any method known in the art for the synthesis of nucleicacid molecules. These include techniques for chemically synthesizingoligonucleotides such as solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro and in vivotranscription of DNA sequences encoding MLF3. Such DNA sequences may beincorporated into a wide variety of vectors with suitable RNA polymerasepromoters such as T7 or SP6. Alternatively, these cDNA constructs thatsynthesize antisense RNA constitutively or inducibly can be introducedinto cell lines, cells, or tissues.

[0131] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0132] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection and by liposomeinjections may be achieved using methods which are well known in theart.

[0133] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0134] An additional embodiment of the invention relates to theadministration of a pharmaceutical composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of MLF3,antibodies to MLF3, mimetics, agonists, antagonists, or inhibitors ofMLF3. The compositions may be administered alone or in combination withat least one other agent, such as stabilizing compound, which may beadministered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones.

[0135] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0136] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable phannaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

[0137] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0138] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0139] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0140] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with a filler or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0141] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as HANKS's solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0142] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0143] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0144] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

[0145] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of MLF3, such labeling wouldinclude amount, frequency, and method of administration.

[0146] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0147] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0148] A therapeutically effective dose refers to that amount of activeingredient, for example MLF3 or fragments thereof, antibodies of MLF3,agonists, antagonists or inhibitors of MLF3, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.

[0149] Pharmaceutical compositions which exhibit large therapeuticindices are preferred. The data obtained from cell culture assays andanimal studies is used in formulating a range of dosage for human use.The dosage contained in such compositions is preferably within a rangeof circulating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0150] The exact dosage will be determined by the practitioner, in lightof factors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

[0151] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

Diagnostics

[0152] In another embodiment, antibodies which specifically bind MLF3may be used for the diagnosis of conditions or diseases characterized byexpression of MLF3, or in assays to monitor patients being treated withMLF3, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for MLF3 includemethods which utilize the antibody and a label to detect MLF3 in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

[0153] A variety of protocols including ELISA, RIA, and FACS formeasuring MLF3 are known in the art and provide a basis for diagnosingaltered or abnormal levels of MLF3 expression. Normal or standard valuesfor MLF3 expression are established by combining body fluids or cellextracts taken from normal mammalian subjects, preferably human, withantibody to MLF3 under conditions suitable for complex formation. Theamount of standard complex formation may be quantified by variousmethods, but preferably by photometric means. Quantities of MLF3expressed in subject, control, and disease samples from biopsied tissuesare compared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

[0154] In another embodiment of the invention, the polynucleotidesencoding MLF3 are used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, antisense RNA andDNA molecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofMLF3 may be correlated with disease. The diagnostic assay may be used todistinguish between absence, presence, and excess expression of MLF3,and to monitor regulation of MLF3 levels during therapeuticintervention.

[0155] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding MLF3 or closely related molecules, may be used to identifynucleic acid sequences which encode MLF3. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5′ regulatory region, or a less specific region,e.g., especially in the 3′ coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding MLF3, alleles, or related sequences.

[0156] Probes may also be used for the detection of related sequences,and should preferably contain at least 50% of the nucleotides from anyof the MLF3 encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO:2 or from genomic sequence including promoter, enhancerelements, and introns of the naturally occurring MLF3.

[0157] Means for producing specific hybridization probes for DNAsencoding MLF3 include the cloning of nucleic acid sequences encodingMLF3 or MLF3 derivatives into vectors for the production of mRNA probes.Such vectors are known in the art, commercially available, and may beused to synthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as ³²P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

[0158] Polynucleotide sequences encoding MLF3 may be used for thediagnosis of conditions or diseases which are associated with expressionof MLF3. Examples of such conditions or diseases include hematopoieticmalignancies and, breast, brain, stomach and paraganglionic tumors. Thepolynucleotide sequences encoding MLF3 may be used in Southern ornorthern analysis, dot blot, or other membrane-based technologies; inPCR technologies; or in dip stick, pin, ELISA or chip assays utilizingfluids or tissues from patient biopsies to detect altered MLF3expression. Such qualitative or quantitative methods are well known inthe art.

[0159] In a particular aspect, the nucleotide sequences encoding MLF3provide the basis for assays that detect activation or induction ofvarious cancers, particularly those mentioned above; in addition thelack of expression of MLF3 may be detected using the MLF3-encodingnucleotide sequences disclosed herein. The nucleotide sequences encodingMLF3 may be labeled by standard methods, and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the biopsied or extracted sample issignificantly altered from that of a comparable control sample, thenucleotide sequences have hybridized with nucleotide sequences in thesample, and the presence of altered levels of nucleotide sequencesencoding MLF3 in the sample indicates the presence of the associateddisease. Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regimen in animal studies, in clinicaltrials, or in monitoring the treatment of an individual patient.

[0160] In order to provide a basis for the diagnosis of diseaseassociated with expression of MLF3, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, which encodes MLF3, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject values is used to establish the presence ofdisease.

[0161] Once disease is established and a treatment protocol isinitiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in the normal patient. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0162] With respect to cancer, the presence of a relatively low or arelatively high amount of transcript in biopsied tissue from anindividual may indicate a predisposition for the development of thedisease, or may provide a means for detecting the disease prior to theappearance of actual clinical symptoms. A more definitive diagnosis ofthis type may allow health professionals to employ preventative measuresor aggressive treatment earlier thereby preventing the development orfurther progression of the cancer.

[0163] Additional diagnostic uses for oligonucleotides designed from thesequences encoding MLF3 may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced from arecombinant source. Oligomers will preferably consist of two nucleotidesequences, one with sense orientation (5′->3′) and another withantisense (3′<-5′), employed under optimized conditions foridentification of a specific gene or condition. The same two oligomers,nested sets of oligomers, or even a degenerate pool of oligomers may beemployed under less stringent conditions for detection and/orquantitation of closely related DNA or RNA sequences.

[0164] Methods which may also be used to quantitate the expression ofMLF3 include radiolabeling or biotinylating nucleotides, coamplificationof a control nucleic acid, and standard curves onto which theexperimental results are interpolated (Melby, P. C. et al. (1993) J.Immunol. Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.229-236). The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor calorimetric response gives rapid quantitation.

[0165] In another embodiment of the invention, the nucleic acidsequences which encode MLF3 may also be used to generate hybridizationprobes which are useful for mapping the naturally occurring genomicsequence. The sequences may be mapped to a particular chromosome or to aspecific region of the chromosome using well known techniques . Suchtechniques include FISH, FACS, or artificial chromosome constructions,such as yeast artificial chromosomes, bacterial artificial chromosomes,bacterial PI constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

[0166] FISH (as described in Verma et al. (1988) Human Chromosomes: AManual of Basic Techniques, Pergamon Press, New York, N.Y.) may becorrelated with other physical chromosome mapping techniques and geneticmap data. Examples of genetic map data can be found in the 1994 GenomeIssue of Science (265:198 If). Correlation between the location of thegene encoding MLF3 on a physical chromosomal map and a specific disease,or predisposition to a specific disease, may help delimit the region ofDNA associated with that genetic disease. The nucleotide sequences ofthe subject invention may be used to detect differences in genesequences between normal, carrier, or affected individuals.

[0167] In situ hybridization of chromosomal preparations and physicalmapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localized by genetic linkage toa particular genomic region, for example, AT to 11q22-23 (Gatti, R. A.et al. (1988) Nature 336:577-580), any sequences mapping to that areamay represent associated or regulatory genes for further investigation.The nucleotide sequence of the subject invention may also be used todetect differences in the chromosomal location due to translocation,inversion, etc. among normal, carrier, or affected individuals,particularly translocations and deletions associated with hematopoieticmalignancies (e.g., AML and myelodysplastic syndrome).

[0168] In another embodiment of the invention, MLF3, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenMLF3 and the agent being tested, may be measured.

[0169] Another technique for drug screening which may be used providesfor high throughput screening of compounds having suitable bindingaffinity to the protein of interest as described in published PCTapplication WO84/03564. In this method, as applied to MLF3 large numbersof different small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with MLF3, or fragments thereof, and washed. Bound MLF3 is thendetected by methods well known in the art. Purified MLF3 can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

[0170] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding MLF3specifically compete with a test compound for binding MLF3. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with MLF3.

[0171] In additional embodiments, the nucleotide sequences which encodeMLF3 may be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

[0172] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0173] I BRAITUT02 cDNA Library Construction

[0174] The BRAITUT02 cDNA library was constructed using 1 microgram ofpolyA RNA isolated from brain tumor tissue removed from the frontal lobeof a 58-year-old Caucasian male during excision of a cerebral meningeallesion. Pathology indicated a grade 2 metastatic hypernephroma. Thepatient presented with migraine headache, developed a cerebralhemorrhage and pulmonary edema, and died during hospitalization. Patienthistory included a grade 2 renal cell carcinoma, insomnia, and chronicairway obstruction. Previous surgeries included a nephroureterectomy.Patient medications included Decadron (dexamethasone) and Dilantin(phenytoin).

[0175] The frozen tissue was homogenized and lysed using a BrinkmannHomogenizer Polytron PT-3000 (Brinkmann Instruments, Westbury N.J.). Thelysate was centrifuged over a 5.7 M CsCl cushion using a Beckman SW28rotor in a Beckman L8-70M Ultracentrifuge (Beckman Instruments) for 18hours at 25,000 rpm at ambient temperature. The RNA was extracted withphenol chloroform pH 4.0, precipitated using 0.3 M sodium acetate and2.5 volumes of ethanol, resuspended in RNAse-free water and DNasetreated at 37 ° C. Extraction and precipitation were repeated as above.RNA was isolated with the QIAGEN OLIGOTEX kit (QIAGEN Inc; ChatsworthCalif.) and used to construct the cDNA library.

[0176] The RNA was handled according to the recommended protocols in theSuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (Cat.#18248-013; Gibco/BRL, Gaithersburg, Md.). cDNAs were fractionated on aSEPHAROSE CL4B column (Cat. #275105, Pharmacia), and those cDNAsexceeding 400 bp were ligated into PSPORT I. The plasmid PSPORT I wassubsequently transformed into DH5α™ competent cells (Cat. #18258-012,Gibco/BRL).

[0177] II Isolation and Sequencing of cDNA Clones

[0178] Plasmid DNA was released from the cells and purified using theMiniprep Kit (Catalog #77468; Advanced Genetic Technologies Corporation,Gaithersburg Md.). This kit consists of a 96-well block with reagentsfor 960 purifications. The recommended protocol was employed except forthe following changes: 1) the 96 wells were each filled with only 1 mlof sterile Terrific Broth (Catalog #22711, Gibco/BRL) with carbenicillinat 25 mg/L and glycerol at 0.4%; 2) the bacteria were cultured for 24hours after the wells were inoculated and then lysed with 60 μl of lysisbuffer; 3) a centrifugation step employing the Beckman GS-6R rotor at2900 rpm for 5 minutes was performed before the contents of the blockwere added to the primary filter plate; and 4) the optional step ofadding isopropanol to TRIS buffer was not routinely performed. After thelast step in the protocol, samples were transferred to a Beckman 96-wellblock for storage.

[0179] The cDNAs were sequenced by the method of Sanger F and A RCoulson (1975; J Mol Biol 94:441f), using a Hamilton MICRO LAB 2200(Hamilton, Reno Nev.) in combination with four Peltier Thermal Cyclers(PTC200 from MJ Research, Watertown Mass.) and Applied Biosystems 377 or373 DNA Sequencing Systems, and the reading frame was determined.

[0180] III Homology Searching of cDNA Clones and Their Deduced Proteins

[0181] Nucleotide or deduced amino acid sequences were used as querysequences against databases such as GenBank, SwissProt, BLOCKS, and PimaII. These databases which contain previously identified and annotatedsequences were searched for regions of homology (similarity) using BLAST(Basic Local Alignment Search Tool; Altschul, S. F. (1993) J. Mol. Evol.36:290-300; Altschul, S. F. et al. (1990) J. Mol. Evol. 215:403-410).

[0182] BLAST produces alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologs which may be of prokaryotic(bacterial) or eukaryotic (animal, fungal or plant) origin. Otheralgorithms such as the one described in Smith R F and T F Smith (1992Protein Engineering 5:35-51), incorporated herein by reference, can beused when dealing with primary sequence patterns and secondary structuregap penalties. As disclosed in this application, the sequences havelengths of at least 49 nucleotides, and no more than 12% uncalled bases(where N is recorded rather than A, C, G, or T).

[0183] The BLAST approach, as detailed in Karlin and Altschul (1993;Proc. Natl. Acad. Sci. USA 90:5873-5877) and incorporated herein byreference, searches matches between a query sequence and a databasesequence, to evaluate the statistical significance of any matches found,and to report only those matches which satisfy the user-selectedthreshold of significance. In this application, threshold was set at10⁻²⁵ for nucleotides and 10⁻¹⁴ for peptides.

[0184] Incyte nucleotide sequences were searched against the GenBankdatabases for primate (pri), rodent (rod), and mammalian sequences(mam), and deduced amino acid sequences from the same clones aresearched against GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp) and eukaryote (eukp), for homology. The relevantdatabase for a particular match were reported as a GIxxx±p (where xxx ispri, rod, etc. and if present, p=peptide).

[0185] A comparison of the full-length and partial cDNA sequences andthe deduced amino acid sequences corresponding to the human MLF3 geneand MLF3 protein with known nucleotide and protein sequences in GenBankrevealed that the full-length human MLF3 cDNA and protein sequences(i.e., SEQ ID NOS:1 and 2) were unique (i.e., not previouslyidentified). This search revealed that the human MLF3 protein sharedsome homology with the human MLF1 protein (SEQ ID NO:3) and the humanMLF2 protein (SEQ ID NO:5). In addition, portions of the amino acidsequence of MLF3 were found to share homology with a number of short ESTsequences of human origin (GI 649669, GI 878231, GI 698722, GI 946359and GI 876176). The five sequences producing the highest-scoring segmentpairs from the GenBank search are shown below in Table 1. In Table 1,“EST” is an abbreviation for expressed sequence tag.; the columndesignations in Table 1 below are as described above. TABLE 1 AccessionDatabase No. Score P(N) Release Description GIpri g1399745 1232 3.6e-166 genpept97 MLF2 GIpri g1066392 354 9.6e-50 genpept97 MLF1 GIprig649669 321 3.0e-54 gb97merck EST GIpri g878231 312 3.7e-37 gb97merckEST GIpri g698722 267 2.7e-30 gb97merck EST

[0186] IV Northern Analysis

[0187] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound (Sambrook et al., supra).

[0188] Analogous computer techniques using BLAST (Altschul, S. F. 1993and 1990, supra) are used to search for identical or related moleculesin nucleotide databases such as GenBank or the LIFESEQ™ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

[0189] The basis of the search is the product score which is defined as:$\frac{\text{\% sequence identity} \times \text{\% maximum BLAST score}}{100}$

[0190] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

[0191] The results of northern analysis are reported as a list oflibraries in which the transcript encoding MLF3 occurs. Abundance andpercent abundance are also reported. Abundance directly reflects thenumber of times a particular transcript is represented in a cDNAlibrary, and percent abundance is abundance divided by the total numberof sequences examined in the cDNA library.

[0192] Electronic northern analysis (FIGS. 4A and 4B) revealed that mRNAencoding human MLF3 (SEQ ID NO:1) was present in libraries generatedfrom a variety of adult and fetal tissues. MLF3 cDNA is most stronglyexpressed in the brain; at least 15% of the libraries containing MFL3sequences were derived from either normal or diseased brain tissue. Inaddition to expression in apparently normal human tissues, MLF3 wasexpressed in a variety of tumors, including but not limited to breast,brain, stomach, and paraganglionic tumors as well as in an immortalizedcell line (the hNT2 teratocarcinoma cell line). MLF3 cDNA was alsoexpressed in a variety of tissues and cell lines which are in thehematopoietic lineages and/or involved with the immune response,including bone marrow, spleen, lymph node, macrophages, and tissuesassociated with inflammatory and/or autoimmune disease (e.g., Crohn'sdisease).

[0193] V Extension of MLF3-Encoding Polynucleotides

[0194] The nucleic acid sequence (SEQ ID NO:2) encoding MLF3 is used todesign oligonucleotide primers for extending a partial nucleotidesequence to full length or for obtaining 5′ or 3′, intron or othercontrol sequences from genomic libraries. One primer is synthesized toinitiate extension in the antisense direction (XLR) and the other issynthesized to extend sequence in the sense direction (XLF). Primers areused to facilitate the extension of the known sequence “outward”generating amplicons containing new, unknown nucleotide sequence for theregion of interest. The initial primers are designed from the cDNA usingOLIGO 4.06 (National Biosciences), or another appropriate program, to be22-30 nucleotides in length, to have a GC content of 50% or more, and toanneal to the target sequence at temperatures about 68°-72° C. Anystretch of nucleotides which would result in hairpin structures andprimer-primer dimerizations is avoided.

[0195] The original, selected cDNA libraries, or a human genomic libraryare used to extend the sequence; the latter is most useful to obtain 5′upstream regions. If more extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

[0196] By following the instructions for the XL-PCR kit (Perkin Elmer)and thoroughly mixing the enzyme and reaction mix, high fidelityamplification is obtained. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR isperformed using the Peltier Thermal Cycler (PTC200; M.J. Research,Watertown, Mass.) and the following parameters:

[0197] Step 1 94° C. for 1 min (initial denaturation)

[0198] Step 2 65° C. for 1 min

[0199] Step 3 68° C. for 6 min

[0200] Step 4 94° C. for 15 sec

[0201] Step 5 65° C. for 1 min

[0202] Step 6 68° C. for 7 min

[0203] Step 7 Repeat step 4-6 for 15 additional cycles

[0204] Step 8 94° C. for 15 sec

[0205] Step 9 65° C. for 1 min

[0206] Step 10 68° C. for 7:15 min

[0207] Step 11 Repeat step 8-10 for 12 cycles

[0208] Step 12 72° C. for 8 min

[0209] Step 13 4° C. (and holding)

[0210] A 5-10 μl aliquot of the reaction mixture is analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products are selected and removedfrom the gel. Further purification involves using a commercial gelextraction method such as QIAQUICK™ (QIAGEN Inc., Chatsworth, Calif.).After recovery of the DNA, Klenow enzyme is used to trimsingle-stranded, nucleotide overhangs creating blunt ends whichfacilitate religation and cloning.

[0211] After ethanol precipitation, the products are redissolved in 13μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1ul T4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 l of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the whole transformationmixture is plated on Luria Bertani (LB)-agar (Sambrook et al., supra)containing 2×Carb. The following day, several colonies are randomlypicked from each plate and cultured in 150 μl of liquid LB/2×Carb mediumplaced in an individual well of an appropriate, commercially-available,sterile 96-well microtiter plate. The following day, 5 μl of eachovernight culture is transferred into a non-sterile 96-well plate andafter dilution 1:10 with water, 5 μl of each sample is transferred intoa PCR array.

[0212] For PCR amplification, 18 μl of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer, andone or both of the gene specific primers used for the extension reactionare added to each well. Amplification is performed using the followingconditions:

[0213] Step 1 94° C. for 60 sec

[0214] Step 2 94° C. for 20 sec

[0215] Step 3 55° C. for 30 sec

[0216] Step 4 72° C. for 90 sec

[0217] Step 5 Repeat steps 2-4 for an additional 29 cycles

[0218] Step 6 72° C. for 180 sec

[0219] Step 7 4° C. (and holding)

[0220] Aliquots of the PCR reactions are run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products arecompared to the original partial cDNAs, and appropriate clones areselected, ligated into plasmid, and sequenced.

[0221] VI Labeling and Use of Hybridization Probes

[0222] Hybridization probes derived from SEQ ID NO:2 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger cDNAfragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmolof each oligomer and 250 μCi of [γ-³²P] adenosine triphosphate(Amersham) and T4 polynucleotide kinase (DuPont NEN®, Boston, Mass.).The labeled oligonucleotides are substantially purified with SEPHADEXG-25 superfine resin column (Pharmacia & Upjohn). A portion containing10⁷ counts per minute of each of the sense and antisenseoligonucleotides is used in a typical membrane based hybridizationanalysis of human genomic DNA digested with one of the followingendonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II; DuPontNEN®).

[0223] The DNA from each digest is fractionated on a 0.7 percent agarosegel and transferred to nylon membranes (Nytran Plus, Schleicher &Schuell, Durham, N.H.). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester, N.Y.) is exposed to the blots in a Phosphoimagercassette (Molecular Dynamics, Sunnyvale, Calif.) for several hours,hybridization patterns are compared visually.

[0224] VII Antisense Molecules

[0225] Antisense molecules to the MLF3-encoding sequence, or any partthereof, is used to inhibit in vivo or in vitro expression of naturallyoccurring MLF3. Although use of antisense oligonucleotides, comprisingabout 20 base-pairs, is specifically described, essentially the sameprocedure is used with larger cDNA fragments. An oligonucleotide basedon the coding sequences of MLF3, as shown in FIGS. 1A and 1B, is used toinhibit expression of naturally occurring MLF3. The complementaryoligonucleotide is designed from the most unique 5′ sequence as shown inFIGS. 1A and 1B and used either to inhibit transcription by preventingpromoter binding to the upstream nontranslated sequence or translationof an MLF3-encoding transcript by preventing the ribosome from binding.Using an appropriate portion of the signal and 5′ sequence of SEQ IDNO:2, an effective antisense oligonucleotide includes any 15-20nucleotides spanning the region which translates into the signal or 5′coding sequence of the polypeptide as shown in FIGS. 1A and 11B.

[0226] VIII Expression of MLF3

[0227] Expression of MLF3 is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector, pSport, previously used for thegeneration of the cDNA library is used to express MLF3 in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

[0228] Induction of an isolated, transformed bacterial strain with IPTGusing standard methods produces a fusion protein which consists of thefirst eight residues of β-galactosidase, about 5 to 15 residues oflinker, and the full length protein or fragments thereof. The signalresidues present on the PSPORT vector direct the secretion of MLF3 intothe bacterial growth media.

[0229] IX Demonstration of MLF3 Activity

[0230] Given the chemical and structural similarity between the humanMLF3 protein and human MLF1 and MLF2 proteins, MLF3 is presumed to be acytoplasmic protein. To demonstrate that MLF3 is a cytoplasmic protein,sequences encoding MLF3 are expressed in cells which lack the ability toexpress MLF3 and the location of MLF3 is ascertained using conventionaltechniques (e.g., immunoprecipitation of proteins derived from cellmembrane-containing fractions and soluble fractions lackingmembrane-associated proteins; preparation of anti-MLF3 antibodies isdescribed below). Expression of MLF3 is achieved using methods known tothe art as described above; numerous expression vectors are availablefor the expression of proteins in eukaryotic and prokaryotic hosts.

[0231] Cells which lack the ability to express human MLF3 are easilyobtained as any non-human eukaryotic cell line is expected to lack theability to express human MLF3; in addition, prokaryotic cells would lackthe ability to express MLF3. Confirmation that a cell lacks the abilityto express MLF3 is obtained by a variety of means known to the artincluding Northern blot analysis in which RNA isolated from thecandidate host cell is hybridized with MLF3-encoding sequences (e.g.,SEQ ID NO:2); cells whose RNA fails to hybridize with MLF3 sequences aresuitable MLF3-negative host cells. In addition, anti-MLF3 antibodies canbe used to confirm that the candidate host cell lacks proteins whichreact or cross-react with MLF3.

[0232] The monkey cell line COS-7 (ATCC CRL 1651) is particularlypreferred when used in conjunction with an expression vector containingthe SV40 origin of replication (SV40 ori) and MFL3 sequences fordetermining the cellular localization of the MLF3 protein. An SV40ori-containing MLF3 expression vector is transferred (e.g.,electroporated) into COS-7 cells and the transiently overexpressed MFL3protein is localized using standard immunohistochemical orimmunofluorescence techniques on fixed an permeabilized transformedCOS-7 cells.

[0233] X Production of MLF3 Specific Antibodies

[0234] MLF3 that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2 is analyzed using DNASTARsoftware (DNASTAR Inc) to determine regions of high immunogenicity and acorresponding oligopolypeptide is synthesized and used to raiseantibodies by means known to those of skill in the art. Selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions, is described by Ausubel et al. (supra), and others.

[0235] Typically, the oligopeptides are 15 residues in length,synthesized using an Applied Biosystems Peptide Synthesizer Model 43 1Ausing fmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH,Sigma, St. Louis, Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel et al.,supra). Rabbits are immunized with the oligopeptide-KLH complex incomplete Freund's adjuvant. The resulting antisera are tested forantipeptide activity, for example, by binding the peptide to plastic,blocking with 1% BSA, reacting with rabbit antisera, washing, andreacting with radioiodinated, goat anti-rabbit IgG.

[0236] XI Purification of Naturally Occurring MLF3 Using SpecificAntibodies

[0237] Naturally occurring or recombinant MLF3 is substantially purifiedby immunoaffinity chromatography using antibodies specific for MLF3. Animmunoaffinity column is constructed by covalently coupling MLF3antibody to an activated chromatographic resin, such as CnBr-activatedSEPHAROSE (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

[0238] Media containing MLF3 is passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of MLF3 (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/MLF3 binding (eg, a buffer of pH 2-3 or a high concentration ofa chaotrope, such as urea or thiocyanate ion), and MLF3 is collected.

[0239] XII Identification of Molecules Which Interact with MLF3

[0240] MLF3 or biologically active fragments thereof are labeled with1251 Bolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled MLF3, washed and any wells withlabeled MLF3 complex are assayed. Data obtained using differentconcentrations of MLF3 are used to calculate values for the number,affinity, and association of MLF3 with the candidate molecules.

[0241] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in molecular biology or related fields are intended to bewithin the scope of the following claims.

1 6 262 amino acids amino acid single linear BRAITUT02 762280 1 Met PheArg Phe Met Arg Asp Val Glu Pro Glu Asp Pro Met Phe Leu 1 5 10 15 MetAsp Pro Phe Ala Ile His Arg Gln His Met Ser Arg Met Leu Ser 20 25 30 GlyGly Phe Gly Tyr Ser Pro Phe Leu Ser Ile Thr Asp Gly Asn Met 35 40 45 ProGly Thr Arg Ala Ala Ser Arg Arg Met Gln Gln Ala Gly Ala Val 50 55 60 XaaPro Phe Gly Xaa Leu Gly Met Ser Gly Gly Phe Met Asp Met Phe 65 70 75 80Gly Met Met Asn Asp Met Xaa Gly Asn Met Glu His Met Thr Ala Gly 85 90 95Gly Asn Cys Gln Thr Phe Ser Ser Ser Thr Val Ile Ser Tyr Ser Asn 100 105110 Thr Gly Asp Gly Ala Pro Lys Val Tyr Gln Glu Thr Ser Glu Met Arg 115120 125 Ser Ala Pro Gly Gly Ile Arg Glu Thr Arg Arg Thr Val Arg Asp Ser130 135 140 Asp Ser Gly Leu Glu Gln Met Ser Ile Gly His His Ile Arg AspArg 145 150 155 160 Ala His Ile Leu Gln Arg Ser Arg Asn His Arg Thr GlyAsp Gln Glu 165 170 175 Glu Arg Gln Asp Tyr Ile Asn Leu Asp Glu Ser GluAla Ala Ala Phe 180 185 190 Asp Asp Glu Trp Arg Arg Glu Thr Ser Arg PheArg Gln Gln Arg Pro 195 200 205 Leu Glu Phe Arg Arg Leu Glu Ser Ser GlyAla Gly Gly Arg Arg Ala 210 215 220 Glu Gly Pro Pro Arg Leu Ala Ile GlnGly Pro Glu Asp Ser Leu Pro 225 230 235 240 Asp Ser Pro Ala Ala Met ThrGly Glu Gly Pro Gly Ala Ser Ala Leu 245 250 255 Leu Tyr Arg Leu Arg Gly260 1322 base pairs nucleic acid single linear BRAITUT02 762280 2GGGGGGCGTA CGGAGGTGGC AGCTGTGGGA GGAGGCGGCG TGGAAGGCCG AGGAGCTCA 60GCCCGGACCA ATCCCCACGT TCCGGGCCGC CACCCTGACC CTGCAGCGTA CCGGGAAG 120AAACCGGCCG GATGGGCCGC TGAGCCCGAA TCGGGCACTG TGTGGAGCCC CCTGGAGC 180AGATCAGGAT GTTCCGCTTC ATGAGGGACG TGGAGCCTGA GGATCCCATG TTCCTGAT 240ATCCCTTTGC TATTCACCGT CAGCATATGA GCCGTATGTT GTCAGGTGGC TTTGGATA 300GCCCCTTCCT CAGCATCACA GATGGCAACA TGCCAGGGAC CAGGGCTGCC AGCCGCCG 360TGCAGCAGGC TGGAGCTGTC TNCCCCTTTG GGNTGCTGGG AATGTCGGGT GGTTTCAT 420ACATGTTTGG GATGATGAAT GACATGNTTG GAAACATGGA ACACATGACA GCTGGAGG 480ATTGCCAGAC CTTCTCATCT TCCACTGTCA TCTCCTACTC CAATACGGGT GATGGTGC 540CCAAGGTCTA CCAAGAGACA TCAGAGATGC GCTCGGCACC AGGCGGGATC CGGGAGAC 600GGAGGACTGT TCGGGATTCA GACAGTGGAC TGGAGCAGAT GTCCATTGGG CATCACAT 660GGGACAGGGC TCACATCCTC CAGCGCTCCC GAAACCATCG CACGGGGGAC CAGGAGGA 720GGCAGGACTA TATCAACCTG GATGAGAGTG AGGCCGCAGC GTTTGATGAC GAGTGGCG 780GGGAGACCTC CCGATTCCGG CAGCAGCGTC CCCTGGAGTT TCGGCGGCTT GAGTCCTC 840GGGCTGGGGG ACGAAGGGCG GAGGGGCCTC CCCGCCTGGC CATCCAGGGA CCTGAGGA 900CCCTTCCCGA CAGTCCCGCC GCTATGACTG GTGAGGGCCC CGGGGCCTCA GCTCTCTT 960ACAGGCTGAG AGGCTGAGAA ATCATCCCCT GAATAACTTT TTCCTCTCGA TTCCCAT 1020CAATTTAATA TTAAATTAAC AGGCAAGCCG GCCCCCACCT CTCCCTGGGG GTCTCAG 1080GAACCTTTCA CGGCACCCTT TCCCTACCTT TTCCTTCTTT AATCTCCTGG TTTACCA 1140ATGACTTCGG CTCTGCATCT ACTTACTTGA TTTTTCATTC TGCCACTTCA TCTTCAA 1200CCCTCACCTT TCCCATCCTA CTCCTGCCAT GCATTGAAGG GTCAATGCAT TTTGGGG 1260GNTTNGGTTT AGGGGCCCCT TCATNCCTNA GCTACCTGGG TCTTTGCCCA ACTTTTC 1320 GA1322 268 amino acids amino acid single linear GenBank 1066392 3 Met PheArg Met Leu Asn Ser Ser Phe Glu Asp Asp Pro Phe Phe Ser 1 5 10 15 GluSer Ile Leu Ala His Arg Glu Asn Met Arg Gln Met Ile Arg Ser 20 25 30 PheSer Glu Pro Phe Gly Arg Asp Leu Leu Ser Ile Ser Asp Gly Arg 35 40 45 GlyArg Ala His Asn Arg Arg Gly His Asn Asp Gly Glu Asp Ser Leu 50 55 60 ThrHis Thr Asp Val Ser Ser Phe Gln Thr Met Asp Gln Met Val Ser 65 70 75 80Asn Met Arg Asn Tyr Met Gln Lys Leu Glu Arg Asn Phe Gly Gln Leu 85 90 95Ser Val Asp Pro Asn Gly His Ser Phe Cys Ser Ser Ser Val Met Thr 100 105110 Tyr Ser Lys Ile Gly Asp Glu Pro Pro Lys Val Phe Gln Ala Ser Thr 115120 125 Gln Thr Arg Arg Ala Pro Gly Gly Ile Lys Glu Thr Arg Lys Ala Met130 135 140 Arg Asp Ser Asp Ser Gly Leu Glu Lys Met Ala Ile Gly His HisIle 145 150 155 160 His Asp Arg Ala His Val Ile Lys Lys Ser Lys Asn LysLys Thr Gly 165 170 175 Asp Glu Glu Val Asn Gln Glu Phe Ile Asn Met AsnGlu Ser Asp Ala 180 185 190 His Ala Phe Asp Glu Glu Trp Gln Ser Glu ValLeu Lys Tyr Lys Pro 195 200 205 Gly Arg His Asn Leu Gly Asn Thr Arg MetArg Ser Val Gly His Glu 210 215 220 Asn Pro Gly Ser Arg Glu Leu Lys ArgArg Glu Lys Pro Gln Gln Ser 225 230 235 240 Pro Ala Ile Glu His Gly ArgArg Ser Asn Val Leu Gly Asp Lys Leu 245 250 255 His Ile Lys Gly Ser SerVal Lys Ser Asn Lys Lys 260 265 1116 base pairs nucleic acid singlelinear GenBank 1066391 4 GTTATGTGTT CCCGTCCGTA CTGGAGGCTA GCTCTTGTCGCGGCCGCGGC GAGTTAACA 60 CGTTTTTCCA ATCTGTCCGC GGCTGCCGCC ACCCAAGACAGAGCCAGAAT GTTCAGGA 120 CTGAACAGCA GTTTTGAGGA TGACCCCTTC TTCTCTGAGTCCATTCTTGC ACACCGAG 180 AATATGCGAC AGATGATAAG AAGTTTTTCT GAACCCTTTGGAAGAGACTT GCTCAGTA 240 TCTGATGGTA GAGGGAGAGC TCATAATCGT AGAGGACATAATGATGGTGA AGATTCTT 300 ACTCATACAG ATGTCAGCTC TTTCCAGACC ATGGACCAAATGGTGTCAAA TATGAGAA 360 TATATGCAGA AATTAGAAAG AAACTTCGGT CAACTTTCAGTGGATCCAAA TGGACATT 420 TTTTGTTCTT CCTCAGTTAT GACTTATTCC AAAATAGGAGATGAACCGCC AAAGGTTT 480 CAGGCCTCAA CTCAAACTCG TCGAGCTCCA GGAGGAATAAAGGAAACCAG GAAAGCAA 540 AGAGATTCTG ACAGTGGACT AGAAAAAATG GCTATTGGTCATCATATCCA TGACCGAG 600 CATGTCATTA AAAAGTCAAA GAACAAGAAG ACTGGAGATGAAGAGGTCAA CCAGGAGT 660 ATCAATATGA ATGAAAGCGA TGCTCATGCT TTTGATGAGGAGTGGCAAAG TGAGGTTT 720 AAGTACAAAC CAGGACGACA CAATCTAGGA AACACTAGAATGAGAAGTGT TGGCCATG 780 AATCCTGGCT CCCGAGAACT TAAAAGAAGG GAGAAACCTCAACAAAGTCC AGCCATTG 840 CATGGAAGGA GATCAAATGT TTTGGGGGAC AAACTCCACATCAAAGGCTC ATCTGTGA 900 AGCAACAAAA AATAAATAGC CATGCATTTG ATTTGTTTAGTTTTGATTGT TTTAACAG 960 AGTAATGGTG CTGGGTAATA AGCATAAGAC CAATCTCTTGCTGTTAAATC AGTTCTG 1020 TTGGCAACTT TCTTCTGATA TCTGAATGTT CATGAAGGTCCTAGCTTTAT ATTGTCC 1080 TTTTAGGAAT AAAATTTTGA TTTTCAACAA AAAAAA 1116 248amino acids amino acid single linear GenBank 1399745 5 Met Phe Arg PheMet Arg Asp Val Glu Pro Glu Asp Pro Met Phe Leu 1 5 10 15 Met Asp ProPhe Ala Ile His Arg Gln His Met Ser Arg Met Leu Ser 20 25 30 Gly Gly PheGly Tyr Ser Pro Phe Leu Ser Ile Thr Asp Gly Asn Met 35 40 45 Pro Gly ThrArg Pro Ala Ser Arg Arg Met Gln Gln Ala Gly Ala Val 50 55 60 Ser Pro PheGly Met Leu Gly Met Ser Gly Gly Phe Met Asp Met Phe 65 70 75 80 Gly MetMet Asn Asp Met Ile Gly Asn Met Glu His Met Thr Ala Gly 85 90 95 Gly AsnCys Gln Thr Phe Ser Ser Ser Thr Val Ile Ser Tyr Ser Asn 100 105 110 ThrGly Asp Gly Ala Pro Lys Val Tyr Gln Glu Thr Ser Glu Met Arg 115 120 125Ser Ala Pro Gly Gly Ile Arg Glu Thr Arg Arg Thr Val Arg Asp Ser 130 135140 Asp Ser Gly Leu Glu Gln Met Ser Ile Gly His His Ile Arg Asp Arg 145150 155 160 Ala His Ile Leu Gln Arg Ser Arg Asn His Arg Thr Gly Asp GlnGlu 165 170 175 Glu Arg Gln Asp Tyr Ile Asn Leu Asp Glu Ser Glu Ala AlaAla Phe 180 185 190 Asp Asp Glu Trp Arg Arg Glu Thr Ser Arg Phe Arg GlnGln Arg Pro 195 200 205 Leu Glu Phe Arg Arg Leu Glu Ser Ser Gly Ala GlyGly Arg Arg Ala 210 215 220 Glu Gly Pro Pro Arg Leu Ala Ile Gln Gly ProGlu Asp Ser Pro Ser 225 230 235 240 Arg Gln Ser Arg Arg Tyr Asp Trp 2451502 base pairs nucleic acid single linear GenBank 1399744 6 CTCTAAAGGGCAGCTGTGGG AGGAGGCGGC GTGGAAGGCC GAGGAGCTCA AGCCCGGAC 60 AATCCCCACGTTCCGGGCCG CGACCCTGAC CCTGCAGCGT ACCGGGAAGC GAAACCGG 120 GGATGGGCCGCTGAGCCCGA ATCGGGCACT GTGTGGAGCC CCCTGGAGCT GAGATCAG 180 TGTTCCGCTTCATGAGGGAC GTGGAGCCTG AGGATCCCAT GTTCCTGATG GATCCCTT 240 CTATTCACCGTCAGCATATG AGCCGTATGT TGTCAGGTGG CTTTGGATAT AGCCCCTT 300 TCAGCATCACAGATGGCAAC ATGCCAGGGA CCAGGCCTGC CAGCCGCCGG ATGCAGCA 360 CTGGAGCTGTCTCCCCCTTT GGGATGCTGG GAATGTCGGG TGGTTTCATG GACATGTT 420 GGATGATGAATGACATGATT GGAAACATGG AACACATGAC AGCTGGAGGC AATTGCCA 480 CCTTCTCATCTTCCACTGTC ATCTCCTACT CCAATACGGG TGATGGTGCC CCCAAGGT 540 ACCAAGAGACATCAGAGATG CGCTCGGCAC CAGGCGGGAT CCGGGAGACA CGGAGGAC 600 TTCGGGATTCAGACAGTGGA CTGGAGCAGA TGTCCATTGG GCATCACATC CGGGACAG 660 CTCACATCCTCCAGCGCTCC CGAAACCATC GCACGGGGGA CCAGGAGGAG CGGCAGGA 720 ATATCAACCTGGATGAGAGT GAGGCCGCAG CGTTTGATGA CGAGTGGCGG CGGGAGAC 780 CCCGATTCCGGCAGCAGCGT CCCCTGGAGT TTCGGCGGCT TGAGTCCTCA GGGGCTGG 840 GACGAAGGGCGGAGGGGCCT CCCCGCCTGG CCATCCAGGG ACCTGAGGAC TCCCCTTC 900 GACAGTCCCGCCGCTATGAC TGGTGAGGGC CCCGGGCCCT CAGCCTCTCT TGTACAGG 960 GAGAGGCTGAGAAATCATCC CCTGAATAAC TTTTTCCTCT CGATTCCCAT CCCCAAT 1020 ATATTAAATTAACAGGCAAG CCGGCCCCCA CCTCTCCCTG GGGGTCTCAG GGAGAAC 1080 TCACGGCACCCTTTCCCTAC CTTTTCCTTC TTTAATCTCC TGGTTTACCA TTGATGA 1140 CGCCTCTGCATCTACTGACT TGATTTTTCA TTCTGCCACT CCATCTTCAA ACCCCCT 1200 CTTTCCCATCCTACTCCTGC CATGCATTGA AGGGTCAATG CATTTTGGGG TGAGCTC 1260 GTTTAGGGGCCCCCTCCATC CCTCAGCTAC CCTGGATCTT TGCCCACCTC TTCCTCA 1320 CCCCCACTGAGGGGCCGTAG CCCTATCTAG GGCTGTGGAA GGAGCAGACT GGTTCCT 1380 TCTCTCCCTCCTCCTGCCCA CACACATCAA AAGAATCTTC CCTACACCCT TCTCTGC 1440 TATTTTTTGATTTGTGCAAC TTGTAACTAG GTGTTTATGG AATAAAGGAG AATGGAA 1500 AG 1502

What is claimed is:
 1. An isolated polypeptide comprising an amino acidsequence selected from the group consisting of: a) a polypeptidecomprising an amino acid sequence of SEQ ID NO:1, b) a naturallyoccurring polypeptide comprising an amino acid sequence at least 90%identical to an amino acid sequence of SEQ ID NO:1, c) a biologicallyactive fragment of a polypeptide having an amino acid sequence of SEQ IDNO:1, and d) an immunogenic fragment of a polypeptide having an aminoacid sequence of SEQ ID NO:1.
 2. An isolated polypeptide of claim 1,having a sequence of SEQ ID NO:1.
 3. An isolated polynucleotide encodinga polypeptide of claim
 1. 4. An isolated polynucleotide encoding apolypeptide of claim
 2. 5. An isolated polynucleotide of claim 4, havinga sequence of SEQ ID NO:2.
 6. A recombinant polynucleotide comprising apromoter sequence operably linked to a polynucleotide of claim
 3. 7. Acell transformed with a recombinant polynucleotide of claim
 6. 8. Atransgenic organism comprising a recombinant polynucleotide of claim 6.9. A method for producing a polypeptide of claim 1, the methodcomprising: a) culturing a cell under conditions suitable for expressionof the polypeptide, wherein said cell is transformed with a recombinantpolynucleotide, and said recombinant polynucleotide comprises a promotersequence operably linked to a polynucleotide encoding the polypeptide ofclaim 1, and b) recovering the polypeptide so expressed.
 10. A method ofclaim 9, wherein the polypeptide has the sequence of SEQ ID NO:1.
 11. Anisolated antibody which specifically binds to a polypeptide of claim 1.12. An isolated polynucleotide comprising a sequence selected from thegroup consisting of: a) a polynucleotide comprising a polynucleotidesequence of SEQ ID NO:2, b) a naturally occurring polynucleotidecomprising a polynucleotide sequence at least 90% identical to apolynucleotide sequence of SEQ ID NO:2, c) a polynucleotide having asequence complementary to a polynucleotide of a), d) a polynucleotidehaving a sequence complementary to a polynucleotide of b) and e) an RNAequivalent of a)-d).
 13. An isolated polynucleotide comprising at least60 contiguous nucleotides of a polynucleotide of claim
 12. 14. A methodfor detecting a target polynucleotide in a sample, said targetpolynucleotide having a sequence of a polynucleotide of claim 12, themethod comprising: a) hybridizing the sample with a probe comprising atleast 20 contiguous nucleotides comprising a sequence complementary tosaid target polynucleotide in the sample, and which probe specificallyhybridizes to said target polynucleotide, under conditions whereby ahybridization complex is formed between said probe and said targetpolynucleotide or fragments thereof, and b) detecting the presence orabsence of said hybridization complex, and, optionally, if present, theamount thereof.
 15. A method of claim 14, wherein the probe comprises atleast 60 contiguous nucleotides.
 16. A method for detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 12, the method comprising: a) amplifyingsaid target polynucleotide or fragment thereof using polymerase chainreaction amplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 17. A composition comprising a polypeptideof claim 1 and a pharmaceutically acceptable excipient.
 18. Acomposition of claim 17, wherein the polypeptide has an amino acidsequence of SEQ ID NO:1.
 19. A method for treating a disease orcondition associated with decreased expression of functional MLF3,comprising administering to a patient in need of such treatment thecomposition of claim
 17. 20. A method for screening a compound foreffectiveness as an agonist of a polypeptide of claim 1, the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting agonist activity in the sample.
 21. Acomposition comprising an agonist compound identified by a method ofclaim 20 and a pharmaceutically acceptable excipient.
 22. A method fortreating a disease or condition associated with decreased expression offunctional MLF3, comprising administering to a patient in need of suchtreatment a composition of claim
 21. 23. A method for screening acompound for effectiveness as an antagonist of a polypeptide of claim 1,the method comprising: a) exposing a sample comprising a polypeptide ofclaim 1 to a compound, and b) detecting antagonist activity in thesample.
 24. A composition comprising an antagonist compound identifiedby a method of claim 23 and a pharmaceutically acceptable excipient. 25.A method for treating a disease or condition associated withoverexpression of functional MLF3, comprising administering to a patientin need of such treatment a composition of claim
 24. 26. A method ofscreening for a compound that specifically binds to the polypeptide ofclaim 1, the method comprising: a) combining the polypeptide of claim 1with at least one test compound under suitable conditions, and b)detecting binding of the polypeptide of claim 1 to the test compound,thereby identifying a compound that specifically binds to thepolypeptide of claim
 1. 27. A method of screening for a compound thatmodulates the activity of the polypeptide of claim 1, said methodcomprising: a) combining the polypeptide of claim 1 with at least onetest compound under conditions permissive for the activity of thepolypeptide of claim 1, b) assessing the activity of the polypeptide ofclaim 1 in the presence of the test compound, and c) comparing theactivity of the polypeptide of claim 1 in the presence of the testcompound with the activity of the polypeptide of claim 1 in the absenceof the test compound, wherein a change in the activity of thepolypeptide of claim 1 in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptideof claim
 1. 28. A method for screening a compound for effectiveness inaltering expression of a target polynucleotide, wherein said targetpolynucleotide comprises a polynucleotide sequence of claim 5, themethod comprising: a) exposing a sample comprising the targetpolynucleotide to a compound, under conditions suitable for theexpression of the target polynucleotide, b) detecting altered expressionof the target polynucleotide, and c) comparing the expression of thetarget polynucleotide in the presence of varying amounts of the compoundand in the absence of the compound.
 29. A method for assessing toxicityof a test compound, the method comprising: a) treating a biologicalsample containing nucleic acids with the test compound, b) hybridizingthe nucleic acids of the treated biological sample with a probecomprising at least 20 contiguous nucleotides of a polynucleotide ofclaim 12 under conditions whereby a specific hybridization complex isformed between said probe and a target polynucleotide in the biologicalsample, said target polynucleotide comprising a polynucleotide sequenceof a polynucleotide of claim 12 or fragment thereof, c) quantifying theamount of hybridization complex, and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 30. Adiagnostic test for a condition or disease associated with theexpression of ABBR in a biological sample, the method comprising: a)combining the biological sample with an antibody of claim 11, underconditions suitable for the antibody to bind the polypeptide and form anantibody:polypeptide complex, and b) detecting the complex, wherein thepresence of the complex correlates with the presence of the polypeptidein the biological sample.
 31. The antibody of claim 11, wherein theantibody is: a) a chimeric antibody, b) a single chain antibody, c) aFab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 32. Acomposition comprising an antibody of claim 11 and an acceptableexcipient.
 33. A method of diagnosing a condition or disease associatedwith the expression of MLF3 in a subject, comprising administering tosaid subject an effective amount of the composition of claim
 32. 34. Acomposition of claim 32, wherein the antibody is labeled.
 35. A methodof diagnosing a condition or disease associated with the expression ofMLF3 in a subject, comprising administering to said subject an effectiveamount of the composition of claim
 34. 36. A method of preparing apolyclonal antibody with the specificity of the antibody of claim 11,the method comprising: a) immunizing an animal with a polypeptide havingan amino acid sequence of SEQ ID NO:1, or an immunogenic fragmentthereof, under conditions to elicit an antibody response, b) isolatingantibodies from said animal, and c) screening the isolated antibodieswith the polypeptide, thereby identifying a polyclonal antibody whichbinds specifically to a polypeptide having an amino acid sequence of SEQID NO:1.
 37. An antibody produced by a method of claim
 36. 38. Acomposition comprising the antibody of claim 37 and a suitable carrier.39. A method of making a monoclonal antibody with the specificity of theantibody of claim 11, the method comprising: a) immunizing an animalwith a polypeptide having an amino acid sequence of SEQ ID NO:1, or animmunogenic fragment thereof, under conditions to elicit an antibodyresponse, b) isolating antibody producing cells from the animal, c)fusing the antibody producing cells with immortalized cells to formmonoclonal antibody-producing hybridoma cells, d) culturing thehybridoma cells, and e) isolating from the culture monoclonal antibodywhich binds specifically to a polypeptide having an amino acid sequenceof SEQ ID NO:1.
 40. A monoclonal antibody produced by a method of claim39.
 41. A composition comprising the antibody of claim 40 and a suitablecarrier.
 42. The antibody of claim 11, wherein the antibody is producedby screening a Fab expression library.
 43. The antibody of claim 11,wherein the antibody is produced by screening a recombinantimmunoglobulin library.
 44. A method of detecting a polypeptide havingan amino acid sequence of SEQ ID NO:1 in a sample, the methodcomprising: a) incubating the antibody of claim 11 with a sample underconditions to allow specific binding of the antibody and thepolypeptide, and b) detecting specific binding, wherein specific bindingindicates the presence of a polypeptide having an amino acid sequence ofSEQ ID NO:1 in the sample.
 45. A method of purifying a polypeptidehaving an amino acid sequence of SEQ ID NO:1 from a sample, the methodcomprising: a) incubating the antibody of claim 11 with a sample underconditions to allow specific binding of the antibody and thepolypeptide, and b) separating the antibody from the sample andobtaining the purified polypeptide having an amino acid sequence of SEQID NO:1.
 46. A microarray wherein at least one element of the microarrayis a polynucleotide of claim
 13. 47. A method of generating anexpression profile of a sample which contains polynucleotides, themethod comprising: a) labeling the polynucleotides of the sample, b)contacting the elements of the microarray of claim 46 with the labeledpolynucleotides of the sample under conditions suitable for theformation of a hybridization complex, and c) quantifying the expressionof the polynucleotides in the sample.
 48. An array comprising differentnucleotide molecules affixed in distinct physical locations on a solidsubstrate, wherein at least one of said nucleotide molecules comprises afirst oligonucleotide or polynucleotide sequence specificallyhybridizable with at least 30 contiguous nucleotides of a targetpolynucleotide, and wherein said target polynucleotide is apolynucleotide of claim
 12. 49. An array of claim 48, wherein said firstoligonucleotide or polynucleotide sequence is completely complementaryto at least 30 contiguous nucleotides of said target polynucleotide. 50.An array of claim 48, wherein said first oligonucleotide orpolynucleotide sequence is completely complementary to at least 60contiguous nucleotides of said target polynucleotide.
 51. An array ofclaim 48, wherein said first oligonucleotide or polynucleotide sequenceis completely complementary to said target polynucleotide.
 52. An arrayof claim 48, which is a microarray.
 53. An array of claim 48, furthercomprising said target polynucleotide hybridized to a nucleotidemolecule comprising said first oligonucleotide or polynucleotidesequence.
 54. An array of claim 48, wherein a linker joins at least oneof said nucleotide molecules to said solid substrate.
 55. An array ofclaim 48, wherein each distinct physical location on the substratecontains multiple nucleotide molecules, and the multiple nucleotidemolecules at any single distinct physical location have the samesequence, and each distinct physical location on the substrate containsnucleotide molecules having a sequence which differs from the sequenceof nucleotide molecules at another distinct physical location on thesubstrate.
 56. A polypeptide of claim 1, comprising the amino acidsequence of SEQ ID NO:1.
 57. A polynucleotide of claim 12, comprisingthe polynucleotide sequence of SEQ ID NO:2.